This invention relates to methods for nucleic acid hybridization, and in particular hybridization performed at low nucleic acid concentrations.
Nucleic acid binding proteins, and in particular, single-stranded DNA binding proteins have been identified in a number of biological systems. Some such proteins play a role in renaturation of DNA. For example, Alberts and Frey, 227Nature 1313, 1970, describe renaturation of purified DNA by the T4 bacteriophage gene 32 protein and propose that it accelerates renaturation by "forcing DNA single strands into an unfolded conformation which leaves their bases available for pairing during chance collisions between complementary strands." Christiansen and Baldwin, 115J. Mol. Biol. 441, 1977, describe reassociation of DNA in the presence of a DNA binding protein from E. coli. They note that spermidine and putrescine together also increase the rate of reassociation of DNA. Weinstock et al., 76 Proc. Natl. Acad. Sci. USA 126, 1979, McEntee, 24 Biochem. 4345, 1985, and Brian and Lehman, 82 Proc. Natl. Acad. Sci. USA 297, 1985, describe DNA renaturation in the presence of recA protein, and its stimulation by ATP and magnesium ions.
Araki and Ogawa, 111 Virology 509, 1981, describe a T7 phage-encoded factor which facilitates binding of nicked open circular ColE1 DNA to cellulose bound ColE1 DNA in the presence of a T7 5'-exonuclease.
Dunn and Studier, 148 J. Mol. Biol. 303, 1981, describe the nucleotide sequence of bacteriophage T7 DNA including a sequence encoding the gene 2.5 protein, which is identified as a single-stranded DNA binding protein. The sequence of this single-stranded DNA binding protein is compared to that from E. coli by Argoss et al., 149 Virology 208, 1986. Significant homology between the two proteins exists.
Scherzinger et al., 123 Mol. General Genetics 247, 1973, Reuben and Gefter, 70 Proc. Natl. Acad. Sci. USA 1846, 1973, and Reuben and Gefter 249 J. Biol. Chem. 3843, 1974, Nakai and Richardson, 263 J. Biol. Chem. 9831, 1988, Fuller and Richardson, 260 J. Biol. Chem. 3197, 1985, Mendelman and Richardson, 266 J. Biol. Chem. 23, 240, 1991, Scherzinger and Klotz, 141 Molec. Gen. Genetics 233, 1975, Araki and Ogawa, 183 Molec. Gen. Genetics 66, 1981, and Araki and Ogawa, 118 Virology 260, 1982 describe properties of gene 2.5 protein. It is identified as having a molecular weight of about 25,000 to 31,000 and as having single-stranded DNA binding properties. It also stimulates DNA synthesis by T7 DNA polymerase on single-stranded templates.